Differential responses of mitotic spindle pole formation to microtubule-stabilizing agents epothilones A and B at low concentrations.

We previously reported that the microtubule-stabilizing agent docetaxel induced formation of fragile acentrosomal spindle poles but that structurally related paclitaxel did not. In the present study, we examined whether the microtubule-stabilizing agents epothilones A and B, which are structurally similar, affect the centrosome/spindle pole architecture.We investigated mitotic processes in epothilone A or B-treated human MDA-MB-435 cells, in which the centrosomes, spindle poles and mitotic micro-tubules were simultaneously visualized by GFP-Aurora A kinase. Fluorescence microscopy of metaphase cells indicated that several chromosomes were misaligned away from the metaphase plate when treated with IC(50) concentrations of epothilone A or B (4.5 or 2 nM, respectively), suggesting that microtubule dynamics was impaired. Interestingly, epothilone B induced formation of acentro-somal spindle poles, but this effect was not observed for epothilone A. Live-cell imaging showed that aster-like structures ectopically arose around the nuclear envelope at the onset of mitosis in epothilone B-treated cells and that one of these asters became an acentrosomal spindle pole. Aster-like structures also arose in the presence of epothilone A, but they were merged into centro-some-derived spindle poles during prometaphase and completely disappeared until metaphase. These results indicate that the centro-some/spindle pole integrity is strongly affected by epothilone B but is not greatly affected by epothilone A. Our findings show that the two epothilones cause different cellular responses at equipotent concentrations and suggest that they have different mechanisms of activity in cells.

A multi-fluorescent MDA435 cell line for mitosis inhibitor studies: simultaneous visualization of chromatin, microtubules, and nuclear envelope in living cells.

As a part of our efforts to create a pre-made multi-fluorescent cell library for various cytological assays, we made a triple-fluorescent cell line in which microtubules, chromosomes, and nuclear envelopes were visualized for simultaneous observation of spindle structure and chromosome distribution in living cells. Pilot experiments with microtubule-disturbing drugs showed the advantages of this cell line in mitosis inhibitor studies.

Live imaging of spindle pole disorganization in docetaxel-treated multicolor cells.

Treatment of cells with docetaxel at low concentrations induces aberrant bipolar spindles of which two centrosomes stay at only one pole, and also induces multipolar spindles. To gain insight into the relations between centrosome impairment and structural defects of the spindle, live-cell imaging was performed on a human MDA Auro/imp/H3 cell line in which centrosomes/mitotic spindles, nuclear membrane and chromatin were simultaneously visualized by fluorescent proteins. In the presence of docetaxel at IC(50) concentration, the centrosomes did not segregate, and multiple aster-like structures ectopically arose around the disappearing nuclear membrane. Those ectopic structures formed an acentrosomal pole opposing to the two-centrosomes-containing pole. In late metaphase, one pole often fragmented into multiple spindle poles, leading multipolar division. These results suggest that spindle pole fragility may be induced by centrosome impairment, and collapse of the pole may contribute to induction of aneuploid daughter cells.